Establishment Of EV-D68 Antigen Capture ELISA

[Background]Enterovirus Type 68(EV-D68),as a viral pathogen isolated from a child hospitalized and manifested pneumonia and bronchiolitis in 1962,is as a member of Picornaviridae family,species Enterovirus D.Although a few of reports about it had been published since then,a large epidemic was observed in 2005.In 2014,a large of epidemic happened in North America since its discovery.This viral Infection usually leads to a series of clinical symptoms including cough,wheezing and hypoxemia in children population.Some report of acute flaccid paralysis cases were thought of being correlated this virus infection.Therefore,it is necessary to establish an ELISA method for rapid detection of EV-D68 as early as possible.[Method]In this study,we used the cell plant to carry out EV-D68 virus amplification after large-scale culture of Vero cells,so as to get more virus stock solution.Concentration was carried out using polyethylene glycol(PEG8000)with molecular weight of 8000.After extraction,iodized cetyl alcohol was used for density gradient centrifugation to obtain a relatively pure antigen,followed by immunization of rabbits.Every three days,taked blood,and specific antibody in serum was measured.When the higher levels animals antibody were tested,sacrificed,separation of serum.Purification of serum IgG was carried out by affinity chromatography.After identification of the product by SDS-PAGE,part of the product was labeled with biotin as the detection antibody and the remaining biotinylated antibody was used as the capture antibody.[Results]Using the matrix method,EV-D68 virus was detected by using the above-mentioned capture antibody and detection antibody.EV-A71 virus solution and Vero cell cryopreservation solution were selected by negative control.Constantly optimize the amount of coated antibody,incubation time,detection of antibody concentration and avidin concentration.Finally,an ELISA system was established to detect enterovirus 68.[Conclusion]According to the relevant provisions of the Chinese Pharmacopoeia,the coefficient of variation and stability of the ELISA method were measured,and the sensitivity and specificity were measured.Finally,an ELISA method for the detection of EV-D68 virus was established.The future EV-D68 virus standardized assay kit provides a strong foundation.